Ion channels are protein molecules on the cell membrane that help transport ions into and out of the cell. To accomplish the mission, ion channels consist of [unreadable]pores[unreadable] through which ions move to cross the membrane. The opening and closing of the channel pore, a process called [unreadable]gating[unreadable], determines if ions can go through the channel, and therefore is critical for controlling many physiological processes. Our long-term interest is to understand how ion channels can function as nano-machines to transport ions across cell membranes. In this application, I propose to continue our work in studying the mechanisms of gating in [unreadable]CLC channels[unreadable], which are important for Cl- transport in a variety of tissues such as skeletal muscle, kidney and brain. Disruptions of these channels, many of which cause malfunctions of channel gating, result in myotonia, kidney diseases, and developmental deficits in brain structures, to name a few. In this proposal, the molecular motions of the CLC channel proteins associated with two gating mechanisms, the [unreadable]fast-gating[unreadable] and the [unreadable]slow/common gating[unreadable] will be studied. The fast-gating controls the opening and closing of the pore with averaged transition time on the order of ~10 ms. Recent studies on the blockade of the pore of CLC-0, a prototype CLC channel, by a chemical compound, parachlorophenoxy acetate (CPA), suggested a conformational change of the pore during fast-gating. Our recent experiments found that various chemical compounds, including a series of fatty acids (FA), could also block the CLC-0 pore with a similar mechanism. We will study the blockade of CLC-0 by CPA and these various FA blockers to explore the possible gating motion associated with the fast-gating. The slow/common-gating of CLC channels operates with a rate slower than that of the fast gating. We recently found that the slow/common-gating of CLC channels may involve a movement of the C-terminal cytoplasmic region of the channel. In addition, ATP, a ubiquitous molecule in all cells, inhibits CLC-1 through modulating the common-gate, presumably by binding to an ATP-binding site in the C-terminal cytoplasmic region. Physiologically, the inhibition of CLC-1 by ATP is critical for the skeletal-muscle fiber to overcome fatigue. Thus, elucidating where the ATP-binding site is and how ATP binding exerts an inhibitory effect on CLC-1 is important for understanding the skeletal-muscle functions. To examine the gating mechanisms of CLC channels, we will employ electrophysiolgical recording and fluorescence imaging techniques. The results from this study not only will lead to a further understanding on the CLC channel functions but will provide insight for designing molecules that can modulate CLC channels, and eventually for developing drugs in treating diseases resulting from CLC channelopathy.